Ras function was analyzed in two well-defined eukaryotic biological models, one for cell proliferation (Xenopus oocytes) and the other for differentiation (3T3 L1 preadipocytes). Microinjection of p21ras into Xenopus oocytes led to germinal vesicle breakdown (first meiotic cell division) even in the absence of protein synthesis. The injected Ras proteins triggered a cascade of phosphorylations leading to activation of the cdc-2 kinase (MPF), the universal eukaryotic regulator of entry into M phase. Prior to MPF activation, microinjected Ras proteins also caused a fast phosphorylation and subsequent activation of cytosolic ERK kinase, including MAP and S6 kinase. Purified Ras proteins could also activate the cytoplasmic ERK kinases when added to highly concentrated cell-free extracts of untreated oocytes. It was shown that insulin- induced differentiation of 3T3 L1 fibroblasts to adipocytes can be mimicked by expression of transfected ras oncogenes but not tyrosine- kinase oncogenes. Exposure of 3T3 L1 cells to insulin caused formation of the active Ras GTP complex, and expression of dominant negative Ras mutants inhibited insulin-induced differentiation. Insulin stimulation or ras expression in these cells resulted also in quick activation of cytosolic serine-threonine kinases, including ERKs (MAP and S6 kinase) and Raf. Insulin-induced differentiation could be blocked by expression of dominant negative ras and raf mutants. However, only dominant negative Ras could block the activation of ERK kinases. In addition, transforming v-raf did not induce ERK activation in these cells. These results indicate that p21ras is mediating insulin signaling in these cells and that Raf kinases act downstream of Ras, but not upstream of ERK kinases in insulin signaling pathways leading to 3T3 L1 differentiation.